Regulation of translational efficiency by different splice variants of the Disc large 1 oncosuppressor 5′‐UTR

Abstract

Human Disc large (DLG1) has been demonstrated to be involved in the control of cell polarity and maintenance of tissue architecture, and is frequently lost in human tumours. However, the mechanisms controlling DLG1 expression are poorly understood. To further examine the regulation of DLG1 expression, we analysed the 5′ ends of DLG1 transcripts by rapid amplification of cDNA ends polymerase chain reaction. We identified an alternative splicing event in the 5′ region of DLG1 mRNA that generates transcripts with two different 5′ untranslated regions (5′‐UTRs). We show by reporter assays that the DLG1 5′‐UTR containing an alternatively spliced exon interferes with the translation of a downstream open reading frame (ORF). However, no significant differences in mRNA stability among the DLG1 5′‐UTR variants were observed. Sequence analysis of the additional exon present in the larger DLG1 5′‐UTR showed the presence of an upstream short ORF which is lost in the short version of the 5′‐UTR DLG1. By mutagenesis and luciferase assays, we analysed the contribution of this upstream short ORF in reducing translation efficiency, and showed that its disruption can revert, to some extent, the negative regulation of large 5′‐UTR. Using computational modelling we also show that the large DLG1 5′‐UTR isoform forms a more stable structure than the short version, and this may contribute to its ability to repress translation. This represents the first analysis of the 5′ region of the DLG1 transcripts and shows that differential expression of alternatively spliced 5′‐UTRs with different translational properties could result in changes in DLG1 abundance.

Transcriptional and translational mechanisms contribute to regulate the expression of Discs Large 1 protein during different biological processes

“…Moreover, we identified the presence of an alternative splicing event in the 5′-UTR of DLG1 that generates two different transcripts: the short and large variants ( Figure 1A). The exclusion of the alternative spliced exon in the short form increases the efficiency of DLG 1 translation, indicating the participation of the 5′-UTR in the control of DLG1 abundance (Cavatorta et al, 2011). We showed that this event is significant in the regulation of DLG1 levels in transformed cells, in agreement with the concept that many oncogenes and tumour suppressor genes express complex 5′-UTRs that participate during carcinogenesis.…”

“…The alternative DLG1 5′-UTRs are expressed in cell lines from different tissues Our previous analyses of DLG1 expression were mainly focused on epithelial cells (Cavatorta et al, 2011). However, this protein was shown to be expressed in several tissues (Lue et al, 1994) and many reports indicate that DLG1 is also involved in many activities in cells such as lymphocytes and neurons.…”

“…We showed that this event is significant in the regulation of DLG1 levels in transformed cells, in agreement with the concept that many oncogenes and tumour suppressor genes express complex 5′-UTRs that participate during carcinogenesis. In fact, translation initiation is highly sensitive to features found in the 5′-UTR, like the presence of AUG(s) upstream of the main ORF, as identified in the large version of the 5′-UTR DLG1 (Cavatorta et al, 2011;Dikstein, 2012 (Cavatorta et al, 2011). In the short version of 5′-UTR DLG the Exon B is absent.…”

“…The length of each exon is not drawn to scale. (B) Total RNA of the indicated cell lines was extracted and subjected to RT-PCR as described (Cavatorta et al, 2011). Both large and short 5′-UTR forms of DLG1 are shown as upper and lower bands, respectively.…”

Selective Phosphorylation of the Dlg1AB Variant Is Critical for TCR-Induced p38 Activation and Induction of Proinflammatory Cytokines in CD8+ T Cells

“…These domains include three PSD-95/Dlg/ZO-1 (PDZ) domains, an SH3 domain and a catalytically inactive guanylate kinase (GUK) domain common to all membrane-associated GUK (MAGUK) scaffolds, as well as an L27 oligomerization domain, an N-terminal proline rich region and a C-terminal HOOK domain, which are unique to Dlg1. Dlg1 domain structure can be modified by alternative splicing events that are thought to effect Dlg1 stability, localization and function (10)(11)(12)(13)(14)(15).…”